Rewiring STAT signaling from the cell surface with Trikine immunotherapeutics [scRNAseq of IL10 Trikine]

Cytokines dimerize two receptor chains to activate Janus kinases and STAT transcription factors that regulate immune cells but have therapeutic liabilities. We engineered “Trikines” to compel cis formation of three-chain cytokine receptor complexes at the cell surface that induce bespoke STAT transcriptional signaling programs optimized for therapeutic efficacy. Designed Trikines co-activated pSTAT5 and pSTAT3 signatures distinct from any natural cytokines, by assembling trimeric combinations of Interleukin-2 (IL-2), Interleukin-10 (IL-10), and Interleukin-21 (IL-21) receptors. IL-2-based-Trikines restrain terminal differentiation of T cells, promote stemness, and enhance durability of tumor control. Unexpectedly, an IL-10-based Trikine induced immune infiltration into poorly immunogenic tumors, showing striking efficacy in small cell lung cancer and pancreatic cancer models. Trikines obviate the need for cell engineering to customize STAT signatures for immunotherapy. Overall design: F1 mice were inoculated s.c. with 5 × 105 KP1 cells and received i.p. administration of mono-IL-10(10) or IL-10/2-Trikine (trikine, 3 µg functional cytokine per dose) every other day from day 7 to day 19, along with anti-PD-1 antibody (Combo, 200 µg per dose, clone RMP1-14) on days 7, 11, 15, and 19. On day 19, mice were sacrificed and tumors were collected. Tumor-infiltrating leukocytes were enriched by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva), followed by surface marker and DAPI staining (Thermo Fisher Scientific). Live CD45? leukocytes were sorted using a Sony SH800 Cell Sorter (Sony Biotechnology) for Single cell RNA sequencing.